WO2018183447A1 - Méthodes de traitement de maladies néoplasiques - Google Patents

Méthodes de traitement de maladies néoplasiques Download PDF

Info

Publication number
WO2018183447A1
WO2018183447A1 PCT/US2018/024764 US2018024764W WO2018183447A1 WO 2018183447 A1 WO2018183447 A1 WO 2018183447A1 US 2018024764 W US2018024764 W US 2018024764W WO 2018183447 A1 WO2018183447 A1 WO 2018183447A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
hom
macrophage
monocyte
tams
Prior art date
Application number
PCT/US2018/024764
Other languages
English (en)
Inventor
Zhenglun Zhu
Original Assignee
Zhenglun Zhu
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhenglun Zhu filed Critical Zhenglun Zhu
Priority to CN201880021980.XA priority Critical patent/CN110709508A/zh
Priority to JP2020502511A priority patent/JP7289289B2/ja
Priority to EP18774517.9A priority patent/EP3601539A4/fr
Priority to CA3058434A priority patent/CA3058434A1/fr
Publication of WO2018183447A1 publication Critical patent/WO2018183447A1/fr
Priority to US16/583,888 priority patent/US20200023006A1/en
Priority to JP2023057097A priority patent/JP2023082128A/ja

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4614—Monocytes; Macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464499—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • TAMs tumor associated macrophages
  • the M2-like TAMs display the characteristic phenotype with elevated expression of IL-10, IL4, MMP, VEGF, but decreased expression of proinflammatory cytokines and cytotoxic iNOs and ROIs implicated in tumoricidal activities.
  • TAMs also contribute to the suppression of anti-tumor immunity by alternating T cells response and balance in tumor microenvironment. The functional plasticity of TAMs was well recognized. It has been proposed that by converting the pro-tumor M2-like TAMs into the anti-tumor Ml -like phenotype, the TAMs could function as an attractive target for anti-tumor therapy.
  • Tumor-specific and tumor-associated antigens have been widely studied as targets for cancer immunotherapy. Targeting these antigens can potentially minimize off-target toxicities while increasing efficacy. On the other hand, antibodies against these antigens are often not effective on their own.
  • a method of treating a cancer comprises providing a modified macrophage or monocyte that contains an exogenous nucleic acid sequence encoding a Hom-1 polypeptide or a fragment thereof that contains the Hom-1 homeobox domain, wherein the modified macrophage or monocyte expresses the Hom-1 polypeptide or the fragment thereof; and administering the modified macrophage or monocyte to a subject with a cancer.
  • the modified macrophage exhibits an Ml phenotype.
  • the nucleic acid sequence is an mRNA molecule.
  • the exogenous nucleic acid sequence is operably linked to a heterologous or endogenous promoter.
  • the method can further include administering an immune modulator to the subject.
  • described herein is a method of treating a cancer that includes contacting a macrophage or monocyte with one or more agents that induce expression of Hom-1, whereby the expression level of endogenous Hom-1 in the macrophage or monocyte is higher than before the contacting step; and administering the thus contacted macrophage or monocyte and an immune modulator to a subject with a cancer.
  • Also described herein is a method of treating a cancer that comprises contacting a macrophage or monocyte with an agent that induces the expression of an Ml gene or an agent that inhibits the expression of an M2 gene, whereby a macrophage that exhibits an Ml- phenotype is generated; and administering the thus generated macrophage and an immune modulator to a subject with cancer.
  • the immune modulator can be selected from the group consisting of CAR-T cells, immune check-point inhibitors, and antibodies against tumor- specific antigens, tumor-associated antigens, or neoantigens.
  • the neoantigen is CK20.
  • Any of methods disclosed herein can further include, prior to the administering step, detecting a lower level of Hom-1 expression in a tumor-associated macrophage in the subject as compared to a control.
  • Hom-1 -regulated TAMs can be used as a new modality of cancer treatment, either alone, or in combinations with immune modulators such as CAR-T cells, immune check-point inhibitors, or antibodies against tumor- specific antigens, tumor-associated antigens or neoantigens, such as CK20 for colorectal cancers, ME1 for lung cancers, and CDC27 for melanomas.
  • immune modulators such as CAR-T cells, immune check-point inhibitors, or antibodies against tumor- specific antigens, tumor-associated antigens or neoantigens, such as CK20 for colorectal cancers, ME1 for lung cancers, and CDC27 for melanomas.
  • Hom-1 a human homeobox transcriptional factor
  • Hom-1 is an antagonist of the canonical Wnt signaling.
  • a nucleic acid sequence of Hom-1 (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) it encodes are disclosed herein. Positions 91-151 within SEQ ID NO: 2 encompass the homeobox domain.
  • Described herein is a method of treating a cancer in a subject by administering to the subject macrophages that exhibit anti-tumor activities.
  • macrophages that exhibit anti-tumor activities As used herein, unless otherwise specified, the terms "macrophages that exhibit anti-tumor activities," "Ml-like
  • macrophages and “macrophages that exhibit an Ml phenotype” may be used
  • Ml-like macrophages can be produced by (1) increasing Hom-1 expression in macrophages or monocytes, (2) increasing expression of one or more Ml genes in macrophages or monocytes, and/or (3) inhibiting expression of one or more M2 genes in macrophages or monocytes.
  • Monocytes e.g., derived from a subject's peripheral blood
  • Hom-1 expression is both necessary and sufficient for monocyte-to- macrophage differentiation.
  • increasing the expression of Hom-1 in monocytes can drive them to differentiate to macrophages.
  • Macrophages or monocytes can be induced ex vivo to express a higher level of endogenous Hom-1.
  • Various agents or treatments can be used to induce Hom-1 expression, e.g., LPS, cholera toxin (CTX), chemotherapeutic agents, radiation, cytokines (e.g., GM- CSF), phorbol 12-myristate 13-acetate (PMA), and antibodies or RNAi against inhibitors of Hom-1 expression.
  • Modified macrophages or monocytes that exhibit an Ml phenotype can be generated by introducing into the macrophages or monocytes an exogenous mRNA molecule (e.g., a synthetic mRNA molecule) that contains a sequence encoding a Hom-1 polypeptide or a fragment thereof that includes the Hom-1 homeobox domain. Hom-1 expression in the macrophages or monocytes is thus increased transiently to induce an Ml phenotype.
  • the modified macrophages or monocytes can then be administered to a cancer patient.
  • the mRNA molecule can be a chemically-modified mRNA to promote stability of the mRNA and/or translation efficiency.
  • Macrophages or monocytes that have been genetically modified to express an elevated level of Hom-1 can also be used to treat a subject with cancer.
  • the genetically modified macrophages or monocytes can contain a nucleic acid sequence encoding a Hom-1 polypeptide or a fragment thereof that includes the Hom-1 homeobox domain.
  • the nucleic acid sequence is operably linked to a promoter and the modified macrophages or monocytes express the Hom-1 polypeptide or the fragment thereof.
  • modified macrophages or modified monocytes express a sufficiently high level of Hom-1 to exhibit anti-tumor activities and/or an Ml phenotype.
  • Genetically modified macrophages or monocytes can also be generated by introducing extra copies of the Hom-1 gene into the macrophages or monocytes.
  • an expression construct containing a Horn- 1 nucleic acid sequence (encoding a Horn- 1 polypeptide or a fragment thereof that includes the Horn- 1 homeobox domain) operably linked to the endogenous Horn- 1 promoter can be introduced into macrophages or monocytes.
  • the expression level of endogenous Hom-1 in macrophages or monocytes can also be increased by genetically modifying regulatory elements of Horn- 1 expression.
  • one or more negative transcription or translation regulatory elements of Hom-1 can be modified, deleted, or replaced to increase Hom-1 transcript and/or protein level.
  • Genome editing techniques utilizing CRIPR, TALEN, or ZFNs or other techniques known in the art can be used to alter regulatory elements of Horn- 1 expression.
  • a Hom-1 polypeptide or fragment thereof that includes the Hom-1 homeobox domain can also be introduced into macrophages or monocytes by direct peptide delivery.
  • an exogenous expression construct for expressing Hom-1 can be introduced (e.g., stably or transiently transfected into) macrophages or monocytes.
  • the Hom-1 nucleic acid sequence is operably linked to a heterologous (i.e., not a Hom-1 promoter) constitutive or inducible promoter.
  • the Hom-1 nucleic acid sequence is operably linked to an endogenous promoter.
  • Ml -like tumoricidal macrophages can also be generated by inducing expression of Ml genes in macrophages or monocytes.
  • Agents that can induce Ml genes include, but not limited to, LPS, CTX, PMA, GM-SCF, INFy, and chemotherapeutic agents.
  • Macrophages or monocytes can also be genetically modified to express elevated levels of Ml genes.
  • Ml genes include ILlb, IL6, IL12, IL23, TNFa, iNOs, CD40, CD80, CD86, CD68, TLR4, TLR2, IL-1R, MHCII, CCL15, CCL20, CXCL9, CXCL1, and SOCS3.
  • Inhibiting expression of M2 genes in macrophages or monocytes can also produce
  • M2 genes include anti-IL4 agents (e.g., antibodies or RNAi agents), anti-IL13 agents (e.g., antibodies or RNAi agents), antibodies against M2 proteins, and RNAi agents targeting M2 genes.
  • M2 genes include ARG1, MMP9, CCL18, VEGF, IL10, IL4, TGFb, CD163, CD206, CD68.,TLR8, TLR1, MHCII, TGM2, DcoyR, IL-1RII, Yml/2, MMR/CD206, and SR.
  • Heterologous or autologous macrophages or monocytes can be used to generate Ml- like macrophages. If heterologous macrophages or monocytes are used, HLA-matching can be conducted to avoid or minimize host reactions. HLA un-matched macrophages or monocytes may also be used. Autologous macrophages or monocytes can be obtained from a cancer patient using methods known in the art.
  • Immune modulators enhance, inhibit, or modulate one or more components of the immune system.
  • modulators include CAR-T cells, immune check-point inhibitors, or antibodies against tumor- specific antigens, tumor-associated antigens or neoantigens, such as CK20 for colorectal cancers, ME1 for lung cancers, and CDC27 for melanomas.
  • the generated Ml-like macrophages and/or an immune modulator can be any suitable Ml-like macrophages and/or an immune modulator.
  • the macrophages, monocytes, and/or immune modulator can be directly injected at a site or into a tissue (e.g., liver or pancreas) or its surrounding area, where a tumor is found.
  • a tissue e.g., liver or pancreas
  • the subject can be treated with the Ml-like macrophages as often (e.g., every 1 to 30 days) and as many times (e.g., 1-30 times) as needed to treat the cancer.
  • the Ml-like macrophages described herein can also be used in a combination therapy with other cancer treatments such as radiation, chemotherapy, and small molecules drugs.
  • any cancer can be treated using the Ml-like macrophages described herein, particularly cancers associated with TAMs that express a low level of Hom-1.
  • cancers that can be treated with Ml-like macrophages include, but are not limited to, carcinoma and sarcoma such as leukemia, sarcoma, osteosarcoma, lymphomas, melanoma, glioma, glioblastoma, pheochromocytoma, hepatoma, ovarian cancer, skin cancer, testicular cancer, gastric cancer, pancreatic cancer, renal cancer, breast cancer, prostate cancer, colorectal cancer, cancer of head and neck, brain cancer, esophageal cancer, bladder cancer, adrenal cortical cancer, lung cancer, bronchus cancer, thyroid cancer, endometrial cancer, nasopharyngeal cancer, cervical cancer, liver cancer, metastatic cancer, and cancer of unknown primary site.
  • carcinoma and sarcoma such as leukemia, sarcoma, osteosarcoma, lymphomas, melanoma, glioma, glioblastoma, pheochromo
  • Detecting a lower expression level of Hom-1 or an Ml gene or a higher expression level of an M2 gene in macrophages found in the microenvironment of a tissue area as compared to that of a control indicates that the tissue area is a cancer or is at risk of becoming a cancer.
  • a control e.g., a corresponding level in a macrophage in a normal tissue
  • a test cell i.e., a macrophage or monocyte
  • a test compound can be contacted with a test compound and the expression level of (i) Hom-1, (ii) a reporter gene operably linked to Hom-1 promoter, (iii) an Ml gene, (iv) a reporter gene operably linked to an Ml promoter, (v) an M2 gene, or (vi) a reporter gene operably linked to an M2 promoter in the test cell is detected.
  • a test compound that increases the expression level of any of (i)-(iv) , and/or decreases the expression level of (v) or (vi) as compared to a control is a candidate compound for treating cancer.
  • a test compound is added to a co-culture containing a test cell and a cancer sample.
  • a test compound is a candidate compound for treating cancer if it, as compared to a control, (i) increases the expression level of Hom-1, a reporter gene operably linked to a Hom-1 promoter, an Ml gene, or a reporter gene operably linked to an Ml promoter in the test cell, (ii) inhibits a significant decrease of the expression level of Hom-1, a reporter gene operably linked to a Hom-1 promoter, an Ml gene, or a reporter gene operably linked to an Ml promoter in the test cell, or (iii) decreases the expression level of an M2 gene or a reporter gene operably linked to an M2 promoter in the test cell.
  • a test compound that inhibits the cancer sample (e.g., inhibits growth of a cancer cell, kills a cancer cell, or decreases the size of the cancer sample) as compared to a control is a candidate compound for treating cancer.
  • the test cell and the cancer sample can be in direct contact with each other. Alternatively, the test cell and the cancer sample are not in direct contact (e.g., with the use of transwell inserts).
  • the cancer sample can be a sample containing a cancer cell, for example, a cancer tissue sample, a cancer cell isolated from a cancer tissue sample, or a cell of a cancer cell line. Cancer tissue samples can be obtained from surgically dissected specimens from cancer patients. Such cancer tissues samples may contain TAMs.
  • a screening method can also be performed with a cancer tissue sample in the absence of a test cell.
  • a cancer tissue sample can be contacted with a test compound.
  • TAMs can be isolated from the cancer tissue sample and the expression level of Hom- 1, an Ml gene, or an M2 gene in the TAMs can be determined. Alternatively or in addition to, the expression level of Hom-1 in the tissue sample can be determined.
  • a test compound is a candidate compound for treating cancer if it, as compared to a control, (i) increases the expression level of Hom-1 or an Ml gene, (ii) inhibits a significant decrease of the expression level of Hom-1 or an Ml gene, and/or (iii) decreases the expression level of an M2 gene.
  • a test compound that inhibits the cancer tissue sample e.g., decreases the size of the sample
  • a test compound that inhibits the cancer tissue sample is also considered as a candidate compound for treating cancer.
  • Test compounds to be screened e.g., proteins, peptides, peptidomimetics, peptoids, antibodies, RNAi, small molecules, or other drugs
  • Test compounds to be screened can be obtained using a method known in the art.
  • the expression level of Hom-1, an Ml gene, or an M2 gene can be determined at either the mRNA level or at the protein level.
  • Promoter activities can also be measured. Methods of measuring mRNA levels, protein levels, and promoter activities are well known in the art.
  • the test cell can be a macrophage or monocyte.
  • the macrophage can be an Ml macrophage, an M2 macrophage, a tumor- associated macrophage, a tissue macrophage, or a monocyte-derived macrophage.
  • the test cell can also be a monocyte.
  • TAMs was significantly decreased in comparison with macrophages isolated from normal control tissues.
  • ectopic expression of Hom-1 converted TAMs into Ml -like phenotype.
  • Both in vitro and in vivo data showed that Horn- 1 conferred tumoricidal activity to TAMs.
  • Hom-1 -expressing TAMs exerted strong inhibitory effects on the growth of a variety of cancers, suggesting the role of Hom-1 -modulated TAMs as a new modality in the treatment of cancers.
  • TAMs display a protumor M2-like phenotype. See, Bronte and
  • FACS analysis showed that, in comparison to macrophages isolated from normal control mucosa, TAMs expressed significant higher levels of cell surface markers associated with M2 phenotypes, such as the CD68, CD163, CD206. See, Zhang et al. (2013), Eur J Cancer 49, 3320-3334. We found that there was no significant difference in the expression of non-discriminating macrophage marker CD33 in TAMs and control macrophages.
  • TAMs can be induced by LPS to display an Ml phenotype. See, Zhang et al. To determine whether Horn- 1 plays a role in TAM plasticity, we examined Hom-1 expression in TAMs exposed to LPS. We found that Hom-1 expression was significantly elevated in TAMs after being stimulated with LPS. Parallel with the elevated expression of Hom-1 and consistent with prior findings, LPS stimulation of TAMs led to elevated secretion of inflammatory cytokines and cytotoxic iNOs.
  • TAMs were isolated and transfected with a plasmid encoding GPF-Hom- 1 or control GFP. Compared with the control GFP transfected TAMs, TAMs transfected with GFP-Hom-1 displayed characteristics Ml morphology with elongated/fibroblast-like cell shape. FACS analysis showed that surface expressions of Ml markers CD40, CD80, and CD86 were significantly increased in TAMs transfected with GFP-Hom-1.
  • Hom-1 promotes tumoricidal function of TAMs in vivo
  • TAMs or monocytes can be induced to exhibit an Ml phenotype by culturing them in an Ml differentiation media.
  • Ml -differentiated TAMs/monocytes can be infused into NSG mice and inhibit cancer growth in vivo.
  • the effects of Ml differentiated TAMs on tumor growth are abolished by inhibition of Hom-1 expression in these TAMs or monocytes.
  • TAMs have been implicated in oncogenesis of essentially all tumors. Following our studies on TAMs in colon cancer cells, we extended our investigation to other tumor types.
  • TAMs Surgical species of lung, melanoma, esophagus, gastric, and pancreatic cancers were obtained, and TAMs were isolated as described above. Macrophages from corresponding normal tissues of the same patient were obtained. Hom-1 expression in TAMs and tissue macrophages were quantified using real time RT-PCR. Horn- 1 expressions in TAMs of all these tumors were low in comparison to Hom-1 expression in corresponding macrophages from distant normal tissues.
  • GFP or GFP-Hom-1 were transfected into the TAMs. After 48 hours of transfection, GFP positive cells were sorted out and co-cultured with individual tumors. Tumor volumes of all tumors decreased during co-culture with GFP-Hom-1 transfected TAMs but not with the control GFP-transfected TAMs. Our results suggested that Hom-1 could convert TAMs into tumoricidal cells independent of tumor types.
  • Cancer tissues and normal tissues were obtained from surgically dissected specimens from patients in pathology lab. Around 5-10 gram of tissue was collected from each tumor mass, or from normal mucosa at 15 cm away from tumor mass. Patient blood samples were also collected.
  • Lamina limbal mononuclear cells were isolated using previously described techniques with modification (Kamada N, et al, 2008; Pignata C, et al, 1990).
  • dissected fresh mucosa and tumor mass were rinsed in 10-cm Petri dish with Ca 2+ -free and Mg 2+ -free hank's balanced salt solution (HBSS) (life technologies) containing 2% fetal bovine serum (FBS) and 1 mM Dithiothreitol (DTT) (Sigma-Aldrich) to remove mucus.
  • HBSS Ca 2+ -free and Mg 2+ -free hank's balanced salt solution
  • FBS fetal bovine serum
  • DTT Dithiothreitol
  • the mucosa and tumor were cut into 0.5 cm pieces by a razor blade and incubated in 6- well plate with 5 mL HBSS containing 1 mM EDTA (Sigma-Aldrich) at 37°C for 1 hour, then passed through a gray-mesh (100 micron).
  • the flowthrough contains intraepithelial lymphocyte and epithelial cells and was analysis by a flow cytometer.
  • HBSS with Ca 2+ and Mg 2+
  • FBS 1.5 mg/mL Collagenase D
  • Dnase I 0.1 mg/mL Dnase I at 37°C for 1 hour.
  • Digested tissues were passed through a gray-mesh (70 micron) filter. The flowthrough was collected and resuspended in a 40% Percoll solution (Pharmacia), then layered on 60% Percoll, and centrifuged at 2000 rpm for 30 min without brake. LPMCs at the interface were collected. Normal mucosal macrophages and TAMs were purified from LPMCs using EasySepTM Human Monocyte/Macrophage Enrichment kit without CD 16 depletion
  • PBMC Peripheral blood mononuclear cells
  • Human monocytes were purified from PBMCs using EasySepTM Human Monocyte Enrichment kit without CD 16 depletion according to the manufacturer's instructions. Purified cells were cultured in completed RPMI medium with 10 ng/mL of M-CSF (PeproTech). After enrichment, monocytes were cultured in completed RPMI medium with M-CSF for 5 days, cells were used for co-culture system.
  • Phenotypic analysis of TAMs and other lymphocytes was performed using flow cytometry after immunolabeling of cells with fluorescence dye-conjugated antibodies.
  • the following antibodies were used: PE-conjugated anti-CD3 (OKT3), -CD25 (BC96), -CD14 (61D3), -CD68 (eBio Y182A), -CD163 (eBio GH161), -CD206, FITC-conjugated anti-CD4 (RPA-T4), -CD33 (HIM3-4), APC-conjugated anti-CD8 (OKT8), -CD4 (OKT4)
  • Granzyme B was performed with PE-conjugated antibodies following the protocol provided by manufacturer. Isotope control labeling was performed in parallel. Antibodies were diluted as recommended by the supplier. Labeled cells were collected on FACScan flow cytometer with Cell-Quest software (BD Biosciences) and analyzed by FlowJo software. Results are expressed as the percentage of positive cells. Organotypic co-cultures of tumor and macrophages
  • Transwell inserts (0.4 ⁇ pore sized, Costar, Corning) were placed in 12-well polystyrene tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). Mucosa and tumor mass were weighed and washed with lx PBS buffer, plus antibiotics, then cut into 0.5 cm pieces. Around 50 mg of tissues were seeded in the upper compartment of a 12-well transwell and filled with 0.5 mL of RPMI 1640 completed medium. 5 x 10 5 of TAMs were added to the lower compartment at the density of 0.5 million cells/well with no direct cell- tissue contact and filled with 2 mL of PRMI completed medium. The plate incubated at 37°C, 5% CO 2 . 0.5 mL of culture medium were collected for cytokine analysis and fresh medium were added every three days. After two weeks co-culture, low chamber
  • Tumors or normal tissues were fixated in Formalin (Fisher Scientific Company, Kalamazoo, MI).
  • CK20 stainings Dako, Carpinteria, CA, clone Ks20.8, 1:50
  • Haematoxylin/eosin (H&E) stainings were performed. CK20 staining was performed on the Leica Bone III staining platform using Epitope Retrieval 2 for 20 minutes online, and using Bone Polymer Refine detection kit. Microscopic analysis was performed with a Nikon Eclipse Ti fluorescence microscopy. Images were captured at an original magnification of 40 x using a color camera applying the NIS Elements imaging software (Nikon). Brightness and contrast for representative images were adjusted equally among groups.
  • GFP-Hom- 1 Transfection of GFP-Hom- 1 into blood macrophages and TAMs was carried out through lipofectamine 2000 (Life technologies) according to manufacture protocol. 48 hours after transfection, cells were filtered through a 70 um filter for cell sorting. GFP positive cells were sorted by BD FACSAria II under the Baker Bio-Protect Hood in a sterile condition. After sorting, cells were cultured in RPMI 1640 completely medium. Hom-1 knockdown
  • Colon TAMs or human primary monocytes were transfected with Morpholino (MO) antisense oligonucleotides using the Human Monocyte Nucleofector Kit (Lonza,
  • oligonucleotides or a standard control MO oligonucleotides were electroporated with the Nucleofector II Device (Lonza). Cells were then immediately removed from the device and incubated overnight with 1ml pre-warmed Human Monocyte Nucleofector Medium containing 2mM glutamine and 10% FBS. Cells were then re-suspended into complete RPMI medium and treated with appropriate cytokines to induce differentiation into macrophages. All the MO oligonucleotides were ordered from Gene Tools (Philomath, OR).
  • IL- ⁇ , IL-10, TNF-a and IL-12p70 in the supernatants of E. coli LPS- (Sigma- Aldrich) treated blood macrophages or LPS-treated TAMs were quantified using ELISA kits obtained from eBiosciences. Analyses were conducted according to the manufacturer's instructions.
  • Arginase activity was quantified in cell lysates by measuring the production of urea using the QuantiChrom arginase Assay Kit (DARG-200; BioAssays Systems). Nitrite concentrations in culture supematants were determined using Griess reagent kit (Molecular Probes).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Epidemiology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)

Abstract

L'invention concerne une méthode de traitement d'un cancer, la méthode comprenant: l'apport d'un macrophage ou d'un monocyte modifié qui contient une séquence d'acide nucléique exogène codant pour un polypeptide Hom-1 ou un fragment de celui-ci qui contient le domaine de boîte homéotique Hom-1, le macrophage ou le monocyte modifié exprimant le polypeptide Hom-1 ou le fragment de celui-ci; et l'administration du macrophage ou monocyte modifié à un sujet atteint d'un cancer.
PCT/US2018/024764 2017-03-28 2018-03-28 Méthodes de traitement de maladies néoplasiques WO2018183447A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CN201880021980.XA CN110709508A (zh) 2017-03-28 2018-03-28 治疗新生性疾病的方法
JP2020502511A JP7289289B2 (ja) 2017-03-28 2018-03-28 腫瘍性疾患を治療する方法
EP18774517.9A EP3601539A4 (fr) 2017-03-28 2018-03-28 Méthodes de traitement de maladies néoplasiques
CA3058434A CA3058434A1 (fr) 2017-03-28 2018-03-28 Methodes de traitement de maladies neoplasiques
US16/583,888 US20200023006A1 (en) 2017-03-28 2019-09-26 Methods for treating neoplastic diseases
JP2023057097A JP2023082128A (ja) 2017-03-28 2023-03-31 腫瘍性疾患を治療する方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201762477754P 2017-03-28 2017-03-28
US62/477,754 2017-03-28
US201762516401P 2017-06-07 2017-06-07
US62/516,401 2017-06-07

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/583,888 Continuation US20200023006A1 (en) 2017-03-28 2019-09-26 Methods for treating neoplastic diseases

Publications (1)

Publication Number Publication Date
WO2018183447A1 true WO2018183447A1 (fr) 2018-10-04

Family

ID=63676762

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2018/024764 WO2018183447A1 (fr) 2017-03-28 2018-03-28 Méthodes de traitement de maladies néoplasiques

Country Status (6)

Country Link
US (1) US20200023006A1 (fr)
EP (1) EP3601539A4 (fr)
JP (2) JP7289289B2 (fr)
CN (1) CN110709508A (fr)
CA (1) CA3058434A1 (fr)
WO (1) WO2018183447A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2022367285A1 (en) * 2021-10-15 2024-05-02 Zhenglun Zhu Method of cancer treatment

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120183553A1 (en) * 2009-07-15 2012-07-19 Zhenglun Zhu Treatment and diagnosis of immune disorders

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2709309B1 (fr) * 1993-08-25 1995-11-10 Centre Nat Rech Scient Compositions cellulaires, préparation et utilisations thérapeutiques.
TWI494319B (zh) * 2007-02-21 2015-08-01 Oncotherapy Science Inc 表現腫瘤相關抗原之癌症的胜肽疫苗
JP5683793B2 (ja) * 2009-06-03 2015-03-11 ウィンテックポリマー株式会社 電気自動車部品用成形品
WO2014093773A1 (fr) * 2012-12-14 2014-06-19 The Brigham And Women's Hospital, Inc. Procédés et essais associés à la différentiation des macrophages
CA2997912A1 (fr) * 2015-09-09 2017-03-16 Seattle Children's Hospital (dba Seattle Children's Research Institute) Modification genetique de macrophages pour l'immunotherapie

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120183553A1 (en) * 2009-07-15 2012-07-19 Zhenglun Zhu Treatment and diagnosis of immune disorders

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANDREESEN ET AL.: "Adoptive Transfer of Tumor Cytotoxic Macrophages Generate in Vitro from Circulating Blood Monocytes: A New Approach to Cancer Immunotherapy", CANCER RESESARCH, vol. 50, no. 23, 1 December 1990 (1990-12-01), pages 7450 - 7456, XP055552595 *
LUGLI ET AL.: "Differential diagnostic and functional role of the multi-marker phenotype CDX2/CK20/CK7 in colorectal cancer stratified by mismatch repair status", MODERN PATHOLOGY, vol. 21, no. 11, November 2008 (2008-11-01), pages 1403 - 1412, XP055552599 *

Also Published As

Publication number Publication date
CA3058434A1 (fr) 2018-10-04
JP2020512402A (ja) 2020-04-23
EP3601539A4 (fr) 2021-01-13
JP2023082128A (ja) 2023-06-13
JP7289289B2 (ja) 2023-06-09
US20200023006A1 (en) 2020-01-23
EP3601539A1 (fr) 2020-02-05
CN110709508A (zh) 2020-01-17

Similar Documents

Publication Publication Date Title
Cao et al. ER stress-induced mediator C/EBP homologous protein thwarts effector T cell activity in tumors through T-bet repression
EP3374497B1 (fr) Modified macrophages pour l'utilisation dans le traitement du cancer
Huang et al. The primordial differentiation of tumor-specific memory CD8+ T cells as bona fide responders to PD-1/PD-L1 blockade in draining lymph nodes
Weulersse et al. Eomes-dependent loss of the co-activating receptor CD226 restrains CD8+ T cell anti-tumor functions and limits the efficacy of cancer immunotherapy
Cortes et al. RHOA G17V induces T follicular helper cell specification and promotes lymphomagenesis
Jacquemin et al. OX40 ligand contributes to human lupus pathogenesis by promoting T follicular helper response
Wu et al. The long noncoding RNA MALAT1 induces tolerogenic dendritic cells and regulatory T cells via miR155/dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin/IL10 axis
Cheng et al. Inhibition of dendritic cell differentiation and accumulation of myeloid-derived suppressor cells in cancer is regulated by S100A9 protein
JP7373543B2 (ja) ２型自然リンパ球細胞、インターロイキン３３、及び／またはインターフェロン誘導性タンパク質４４による癌免疫の調節
Kudo-Saito et al. Induction of immunoregulatory CD271+ cells by metastatic tumor cells that express human endogenous retrovirus H
Ohtsuka et al. Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells
Bi et al. E-cadherin expression on multiple myeloma cells activates tumor-promoting properties in plasmacytoid DCs
Wang et al. Siglec receptors modulate dendritic cell activation and antigen presentation to T cells in cancer
US20220226379A1 (en) Dnmt3a knock-out stat5 activated genetically engineered t-cells
US20230312671A1 (en) Grp78 targeted adoptive cell therapy
JP2023082128A (ja) 腫瘍性疾患を治療する方法
Fu et al. Glycogen synthase kinase 3 controls T-cell exhaustion by regulating NFAT activation
Que et al. Combinations of anti-GITR antibody and CD28 superagonist induce permanent allograft acceptance by generating type 1 regulatory T cells
WO2024071411A1 (fr) CELLULE IMMUNITAIRE INDUITE À PARTIR D'UNE CELLULE iPS
고재문 The effect of IL-23-producing human lung cancer cells on tumor growth via conversion of innate lymphoid cell 1 (ILC1) into ILC3
Alorro Exploring STAT3 as a Therapeutic Target in Mouse Models of Gastrointestinal Cancer
Sarode Reprogramming of tumor-associated macrophages by targeting β-catenin-FOSL2-ARID5A signaling–novel treatment of lung cancer
Wu Investigation of T-cell Suppression in the Acute Myeloid Leukaemia Microenvironment
Catena et al. Systemic effects of melanoma-secreted MIDKINE in the inhibition of dendritic cell differentiation and function
Jansen et al. Chemotherapy-induced intestinal epithelial damage directly promotes galectin-9-driven modulation of T cell behavior

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18774517

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3058434

Country of ref document: CA

Ref document number: 2020502511

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2018774517

Country of ref document: EP

Effective date: 20191028